The objective was to determine the location of blaCTX-M-1 and blaCMY-2 genes in 33 Escherichia coli isolates previously obtained from healthy humans, pets, and food-producing animals in Tunisia, and to characterize the genetic lineages of isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE)-XbaI and multilocus sequence typing (MLST). Plasmids were analyzed by S1-PFGE, polymerase chain reaction-based replicon typing, and plasmid MLST. Conjugation experiments were performed. The blaCTX-M-1 and blaCMY-2 genes were studied by I-Ceu1-PFGE and S1-PFGE, and subsequent hybridization with specific probes. Eighteen different sequence types (STs) were identified among the 30 CTX-M-1-producing isolates, 5 of them being detected in 17 isolates (ST/phylogroup): ST57/D, ST155/B1, ST58/B1, ST10/A, and ST398/A. Most of the blaCTX-M-1-positive isolates had different PFGE profiles, with the exception of four human and pet isolates of lineage ST57 with related PFGE profiles (>80% identity). Three CMY-2-producing isolates were typed as ST58/B1, ST117/D, and ST3632/B2. The IncI1 replicon was detected in all the 33 E. coli studied isolates, in many cases in combination with other replicons: IncF, IncX, IncK, IncR, IncY, colE, or IncN. The blaCTX-M-1 gene was transferred by conjugation in 22 of the 30 positive strains and was located into IncI1 plasmids (ST3-CC3); the blaCMY-2 gene was located into a conjugative IncI1 plasmid (ST12) of 97 kb in one strain. One blaCTX-M-1-positive strain carried the qnrB19 gene in a 33 kb IncX plasmid. Diverse genetic lineages are detected in extended-spectrum beta-lactamase- and AmpC beta-lactamase-producing E. coli from different origins. The blaCTX-M-1 and blaCMY-2 genes were associated with conjugative IncI1 (ST3 and ST12, respectively) plasmids in E. coli strains from human and animal origin.